Novel genes and proteins encoded thereby

ABSTRACT

A novel DNA comprising a protein-encoding region is directly cloned from cDNA library originating from human adult whole brain and human fetal whole brain, the base sequence thereof is determined, and the functions thereof are identified. A DNA comprising a base sequence encoding the following polypeptide (a) or (b): (a) a polypeptide consisting of an amino acid sequence which is identical or substantially identical with the amino acid sequence represented by SEQ ID No.1, (b) a polypeptide consisting of an amino acid sequence represented by SEQ ID No.1 in which part of amino acids are deleted, substituted or added, and having substantially the same biological activity as the function of the polypeptide (a), recombinant polypeptide encoded by the above DNAs, and protein comprising the polypeptide.

FIELD OF THE INVENTION

The present invention is related to a novel DNA and a gene comprisingthe DNA, a recombinant polypeptide encoded by the DNA and a novelrecombinant protein comprising the polypeptide. More particularly, it isrelated to a novel protein that is supposed to belong to Quiescin Q6family and a gene encoding thereof.

BACKGROUND OF THE INVENTION

A grand scale sequencing in the Human Genome Project has been producingand analyzing a lot of information on the nucleotide sequences of humangenome every day.

A final goal of the project is not only to determine the whole genomicnucleotide sequences, but also to reveal and understand various humanlife phenomena based on the information about their structure, i.e., DNAsequence information.

[Non-Patent Document 1]

Donald L. Coppock, et al., Genomics 54, “The Quiescin Q6 Gene (QSCN 6)is a Fusion of Two Ancient Gene Families: Thioredoxin and ERV1”, 1998,p.460-468

[Non-Patent Document 2]

Beatrice Beayoun, et al., The Journal of Biological Chemistry Vol.276,No.17, “Rat Seminal Vesicle FAD-dependent Sulfhydryl Oxidase”, 2001,p.13830-13837.

Regions encoding proteins occupy only a small part of the human genome.Although the coding region may recently be predicted by utilizingtechniques in information technology such as neural network and hiddenmarkov model, their predictive accuracy is not yet enough.

SUMMARY OF THE INVENTION

The present inventors have succeeded in directly cloning a novel DNAcomprising a region encoding a protein from cDNA library derived fromhuman adult whole brain and human fetal whole brain, and in determiningits nucleotide sequence, and have completed the present invention.

A first aspect of the present invention relates to a DNA comprising anucleotide sequence encoding the following polypeptide (a) or (b):

-   -   (a) a polypeptide consisting of an amino acid sequence which is        identical or substantially identical with an amino acid sequence        represented by SEQ ID No.1,    -   (b) a polypeptide consisting of an amino acid sequence        represented by SEQ ID No.1 in which part of amino acids are        deleted, substituted or added, and having substantially the same        biological activity (function) as the function of the        polypeptide (a).

One example of the above DNA is that comprising a base sequencerepresented by SEQ ID No.1.

A second aspect of the present invention relates to a DNA hybridizingwith a DNA having a base sequence complementary to the DNA of the firstaspect of the present invention under stringent conditions, and havingsubstantially the same biological activity as the function of the abovepolypeptide (a).

The DNAs of the first and second aspects will be also referred to as“the present DNA” in the present specification. The present invention isalso related to an antisense DNA having a base sequence that issubstantially complementary to the present DNA.

A third aspect of the present invention relates to a gene constructcomprising the present DNA. The term “gene construct” in the presentspecification means any gene that is artificially manipulated. The geneconstruction includes, for example, a vector comprising the present DNAor antisense DNA, and an expression vector comprising the present DNA.

A forth aspect of the present invention relates to A polypeptide (a) or(b):

-   -   (a) a polypeptide consisting of an amino acid sequence which is        identical or substantially identical with the amino acid        sequence represented by SEQ ID No.1,    -   (b) a polypeptide consisting of an amino acid sequence        represented by SEQ ID No.1 in which part of amino acids are        deleted, substituted or added, and having substantially the same        biological activity as the function of the above polypeptide        (a).

A fifth aspect of the present invention relates to a recombinantpolypeptide encoded by the gene construct of the third aspect of thepresent invention.

The above polypeptides are also referred to as “the present polypeptide”in the present specification. The term “polypeptide” in thisspecification means a “polymer of amino acids having any molecularweight.” The present invention may contain a recombinant proteincomprising the present polypeptide. As there is no limitation in themolecular weight with respect to the polypeptide of the presentinvention, the present polypeptide may also contain the recombinantprotein.

A sixth aspect of the present invention relates to an antibody againstthe present polypeptide.

A seventh aspect of the present invention relates to a DNA tip on whichthe present DNAs are arrayed.

A eighth aspect of the present invention relates to a polypeptide tip onwhich the present polypeptides are arrayed.

A ninth aspect of the present invention relates to an antibody tip onwhich the antibodies of the sixth aspect of the present invention arearrayed.

A tenth aspect of the present invention relates to an antisenseoligonucleotide to the present DNA.

BEST MODE FOR CARRYING OUT THE INVENTION

The present DNA is isolated as cDNA fragment from a cDNA libraryprepared by the present inventors by using as starting materials mRNAsof human adult whole brain and human fetal whole brain, which arecommercially available from Clontech, and identified with determinationof its nucleotide sequence.

Thus, clones are randomly isolated from the library derived from humanadult whole brain and human fetal whole brain, which is prepared inaccordance with Ohara et al., DNA Research Vol.4, 53-59 (1997).

Homology search is done on databases of known genes with the use of thenucleotide sequences at both ends thus obtained clones as a query tofind new clones. The 5′ and 3′ terminal sequences of the new clones arealigned with the human genome. When an unknown gene with a long basesequence is found, a whole length analysis is done for the unknown gene.

Paying much attention not to make any artificial errors in shortfragments or determined sequences, the whole region of human genescomprising the present DNA may be prepared also by using PCR methodssuch as RACE.

The present invention further relates to a recombinant vector comprisingthe present DNA or the gene construct comprising the present DNA, to atransformant harboring the recombinant vector, to a method for theproduction of the present polypeptide, the recombinant proteincomprising the polypeptide or salts thereof comprising culturing thetransformant, and producing, accumulating and recovering the presentpolypeptide or the recombinant protein comprising the polypeptide, andto the present polypeptide, the recombinant protein comprising thepolypeptide or salts thereof thus obtained.

The present invention still further relates to a pharmaceuticalcomposition comprising the present DNA or gene construct, to apolynucleotide (DNA) encoding the present polypeptide or partialpolypeptide thereof or the recombinant protein comprising thepolypeptide, to the antisense oligonucleotide to the present DNA, to apharmaceutical composition comprising the above polynucleotide or theantisense nucleotide, and to pharmaceutical composition comprising thepresent polypeptide or partial polypeptide thereof or the recombinantprotein comprising the polypeptide.

The present invention also relates to the DNA tip, peptide tip andantibody tip that are prepared by arraying the present DNAs, the presentpolypeptides and the antibodies against the present polypeptides,respectively.

Still the present invention relates to a method for screening of acompound which specifically interact with the present polypeptide orpartial polypeptide thereof, the recombinant protein comprising thepolypeptide, salts thereof or antibodies against them by using thesematerials, a screening kit, and a compound identified by the screeningmethod.

The present DNA may be any DNA as long as it comprises the base sequenceencoding the present polypeptide, including a cDNA identified andisolated from cDNA libraries derived from human brain and other tissuesor cells such as heart, lung, liver, spleen, kidney and testis, and asynthetic DNA.

A vector, which is used in the preparation of the libraries, includesbacteriophage, plasmid, cosmido and phagemid. The cDNA may be alsoamplified by means of Reverse Transcription coupled Polymerase ChainReaction (RT-PCR) with the use of a total RNA or mRNA fraction preparedfrom the above tissues or cells.

The antisense oligonucleotide (DNA) to the present DNA may includes anyantisense DNA having the base sequence that is substantiallycomplementary to the DNA encoding the present polypeptide or the partialpolypeptide thereof, and having a function of inhibiting the expressionof the present DNA.

The “base sequence that is substantially complementary to the presentDNA” means, for example, a base sequence having homology of about 90% ormore, preferably about 95% or more, more preferably about 100% to thewhole or partial base sequence complementary to the present DNA. Anynucleic acid sequence having the function similar to that of the aboveantisense DNA such as a modified RNA or DNA may be included in theantisense oligonucleotide of the present invention. These antisenseoligonucleeotide may be produced by a known DNA synthesizer or the like.

The “amino acid sequence which is substantially identical with an aminoacid sequence represented by SEQ ID No.1” means an amino acid sequencehaving homology on an average of about 70% or more, preferably about 80%or more, more preferably about 90% or more, further more preferablyabout 95% or more to the whole amino acid sequence represented by SEQ IDNo.1.

Thus, a polypeptide consisting of the amino acid sequence which issubstantially identical with the amino acid sequence represented by SEQID No.1 includes a polypeptide having the above homology to the aminoacid sequence represented by SEQ ID No.1 and having substantially thesame biological activity (or function) as the function of a polypeptideconsisting of the above amino acid sequence. The term “substantially thesame” means the activities or functions of the both substances are thesame with each other in quality or property.

The present polypeptide includes a polypeptide consisting of the aminoacid sequence represented by SEQ ID No.1 in which part of amino acids(preferably 1˜20, more preferably 1˜10, further more preferably a fewamino acids) are deleted, substituted or added, and having substantiallythe same biological activity (or function) as the function of thepolypeptide consisting of the amino acid sequence represented by SEQ IDNo.1.

The polypeptide consisting of the amino acid sequence which issubstantially identical with the amino acid sequence represented by SEQID No.1, or the polypeptide consisting of the amino acid sequencerepresented by SEQ ID No.1 in which part of amino acids are deleted,substituted or added may be easily prepared by well known methods suchas site-specific mutation, genetic homologous recombination, primerextension method and PCR, or any optional combinations thereof.

In order for the polypeptide or protein to have substantially the samebiological activity, it is possible to make a substitution among aminoacids belonging to the same group (polar, non-polar, hydrophobic,hydrophilic, positive-charged, negative-charged, or aromatic amino acidgroup) in the amino acids that constitute the present polypeptide.Alternatively, it is desirable to keep amino acids which are included ina functional domain.

The present DNA includes the DNA comprising the base sequence encodingthe polypeptide represented by SEQ ID No.1, and the DNA hybridizing withthe DNA complementary to the DNA of the first aspect under stringentconditions and having substantially the same biological activity as thefunction of the polypeptide consisting of the amino acid sequence whichis identical or substantially identical with the amino acid sequencerepresented by SEQ ID No.1.

The DNA that may hybridizes under the above stringent conditionsincludes a DNA having homology on an average of about 80% or more,preferably about 90% or more, more preferably about 95% or more to thewhole base sequence of the above DNA

Hybridization may be performed in accordance with a method described in,for example, Current protocols in molecular biology (edited by FrederickM. Ausubel et al., 1987). If a commercial library is used it may be doneaccording to a method described in instructions attached thereto.

The phrase “stringent conditions” in this specification means conditionsunder which Southern blot hybridization is carried out in an aqueoussolution containing 1 mM Na EDTA, 0.5M Na₂HPO₄ (pH 7.2) and 7% SDS at65° C., followed by the washing of a membrane with an aqueous solutioncontaining 1 mM Na EDTA, 40 mM Na₂HPO₄ (pH 7.2) and 1% SDS at 65° C. Thesame stringency may be realized by using other conditions.

The present DNA may be cloned by preparing a synthetic DNA primer withan appropriate nucleotide sequence such as a part of the polypeptide ofthe present invention, and amplifying it with an appropriate library bymeans of PCR. The present DNA may be further selected from DNAsintegrated into appropriate vectors by means of hybridization with a DNAfragment or synthetic DNA encoding the whole region or part of thepresent polypeptide.

Hybridization may be performed in accordance with a method described in,for example, Current protocols in molecular biology (edited by FrederickM. Ausubel et al., 1987). If a commercial library is used it may be doneaccording to a method described in instructions attached thereto.

The present DNA thus cloned may be directly used, or optionally digestedwith a restriction enzyme or tagged with a linker for use. The presentDNA may have a translation initiation codon “ATG” at its 5′-end, and atranslation termination codon, “TAA”, “TGA” or “TAG” at its 3′ end.These codons may be also added by using an appropriate synthetic DNAadapter.

The expression vector may be constructed by any known method in the art.For example, it is made by (1) excising a DNA fragment containing thepresent DNA or the gene comprising the DNA, and (2) ligating the DNAfragment downstream of a promoter in the expression vector.

Vectors to be used in the present invention include those derived fromEscherichia coli such as pBR322, pBR325, pUC18, pUC118; those derivedfrom Bacillus subtilis such as pUB110, pTP5 and pC194; those derivedfrom yeast such as pSH19 and pSH15; bacteriophage such as λphage; animalviruses such as retorovirus, vaccinia virus and baculovirus.

Promoters to be used in the present invention may be any promoterssuitable for a host cell which is used in the expression of the gene,including, for example, trp promoter, lac promoter, recA promoter, λPLpromoter and 1 pp promoter for E. coli; SPO1 promoter, SPO2 promoter andpenP promoter for Bacillus subtilis; PHO5 promoter, PGK promoter, GAPpromoter and ADH promoter for yeast; and SRα promoter, SV40 promoter,LTR promoter, CMV promoter and HSV-TK promoter for animal cells.

Other elements known in the art such as an enhancer, a splicing signal,a polyadenylation signal, a selection marker and SV40 replication originmay be added to the expression vectors. The protein encoded by thepresent DNA may be optionally expressed as a fused protein with otherproteins such as glutathione-S-transferase and protein A. The fusedprotein may be cleaved by an appropriate protease and separated intoeach protein.

The host cell used in the present invention includes Escherichia,Bacillus, yeast, insect cells, and animal cells.

The examples of Escherichia include E. coli K-12•DH1 (Proc. Natl. Acad.Sci., USA, vol.60 160 (1968)), JM103 (Nucleic Acids Research, vol.9, 309(1981)), JA221 (Journal of Molecular Biology, vol.120, 517 (1978)) andHB101 (Journal of Molecular Biology, vol.41, 459 (1969)).

The examples of Bacillus include Bacillus subtilis MI114 (Gene vol.24,255 (1983)), and 207-21(Journal of Molecular Biology, vol.95, 87(1984)).

The examples of yeast include Saccaromyces cerevisiae AH22, AH22R-,NA87-11A, DKD-5D, and 20B-12; Schizosaccaromyces pombe NCYC1913,NCYC2036; and Saccaromyces picjia pastoris.

The examples of animal cells include simian cell COS-7, Vero, Chinesehamster cell CHO (“CHO cell”), dhfr gene-defective CHO cell, mouse Lcell, mouse AtT-20 cell, mouse myeloma cell, rat GH3 cell and human FLcell.

The transfomation of these cells may be carried out in accordance with amethod known in the art such as those described in the followingarticles:

Proc. Natl. Acad. Sci., USA vol.69, 2110 (1972); Gene, vol.17,107(1982), Molecular & General Genetics, vol.168, 111 (1979); Methods inEnzymology, vol. 194, 182-187 (1991); Proc. Natl. Acad. Sci., USAvol.75, 1929 (1978); Cell Engineering, additional volume 8, “New CellEngineering experimental protocols, 263-267 (published by Shu-junn Co.);and Virology vol.52 456 (1973).

The transformant thus transformed with the expression vector comprisingthe present DNA or the gene comprising thereof may be cultured accordingto a method known in the art.

Escherichia host cells may be normally cultured at about 15˜43° C. forabout 3˜24 hours with aeration and stirring, if necessary. Bacillus hostcells may be normally cultured at about 30˜40° C. for about 6˜24 hourswith aeration and stirring, if necessary.

Yeast host cells may be normally cultured in a culture medium with pHabout 5˜8 at about 20˜35° C. for about 24f˜72 hours with aeration andstirring, if necessary.

Animal host cells may be normally cultured in a culture medium with pHabout 6˜8 at about 30˜40° C. for about 15˜60 hours with aeration andstirring, if necessary.

The polypeptide or protein according to the present invention may beisolated and purified from the above culture as follows. After thecompletion of culturing, bacteria or cells are collected by a knownmethod, suspended in an appropriate buffer solution, and destroyed bymeans of ultrasonic, lysozyme and/or freezing and thawing treatment,followed by centrifugation or filtration to give a crude proteinextract. The buffer solution may contain a protein-denaturing agent suchas urea and guanidine hydrochloride, or a surfactant such asTritonX-100™. If the protein is secreted into the culture medium, thebacteria or cells are separated from its supernatant by a known methodafter the completion of culturing, and the resulting supernatant iscollected. The protein thus obtained and contained in the culturesupernatant or extract may be purified by an appropriate combination ofknown separation and purification methods.

The present polypeptide or protein thus obtained may be converted intotheir salt form, which may be converted into its free from vice versa orinto other salt forms according to a known method. The protein producedby the transformant may be treated with an appropriate protein-modifyingenzyme such as trypsin or chymotrypsin in order to optionally addmodification to it or to partially remove polypeptide from it before orafter purification.

The presence of present polypeptide or protein or salt thereof may bedetermined by various binding assay methods or enzyme immunoassay usinga specific antibody.

The C-terminus of the polypeptide of the present invention is normally acarboxyl group (—COOH) or a carboxylate (—COO—), but the C terminus maybe an amide (—CONH₂) or ester (—COOR). Examples of R in ester that areused herein include a C1-6 alkyl group, such as methyl, ethyl, n-propyl,isopropyl or n-butyl; a C3-8 cycloalkyl group, such as cyclopentyl orcyclohexyl; a C6-12 aryl group, such as phenyl or α-naphthyl; aphenyl-C1-2 alkyl group, such as benzyl or phenethyl; and a C7-14aralkyl group, such as an α-naphthyl-1-2 alkyl group, e.g., α-naphthylmethyl. Further, pivaloyl-oxymethyl ester generally used as oraladministration may also be used.

When the polypeptide of the present invention has a carboxyl group (orcarboxylate) other than at the C-terminus, the polypeptide of thepresent invention encompasses such a polypeptide wherein the carboxylgroup is amidated or esterified. An example of the ester that is used inthis case is the above-mentioned ester at the C-terminus. Moreover thepolypeptide of the present invention also encompasses a polypeptidewherein an amino group of a methionine residue at the N-terminus isprotected with a protecting group (for example, a C1-6 acyl group, suchas a formyl group or an acetyl group); a polypeptide wherein a glutamicacid residue at the N-teminus which is generated by in vivo cleavage ispyroglutamated; a polypeptide wherein OH, COOH, NH₂, SH and the like onthe side chain of intramolecular amino acids are protected withappropriate protecting groups ( for example, a C1-6 acyl group, such asa formyl group and an acetyl group); or a complex protein, such as aso-called glycoprotein formed by the binding of sugar chains to apolypeptide, or the like.

A partial polypeptide of the polypeptide of the present invention may beany partial peptide of the above-mentioned polypeptide of the presentinvention and has substantially the same activities. For example, apolypeptide that is used herein comprises a sequence of at least 10 ormore, preferably 50 or more, further preferably 70 or more, further morepreferably 100 or more, and most preferably 200 or more amino acids ofthe amino acid sequence of the polypeptide of the present invention,and, for example, has substantially the same biological activity withthe function of the polypeptide of the present invention. An example ofa preferable partial polypeptide of the present invention contains eachfunctional domain. Further, the partial peptide of the present inventionnormally has a carboxyl group (—COOH) or a carboxylate (—COO—) at theC-terminus, and it may also have an amide (—CONH₂—) or an ester (—COOR)at the C-terminus like the above polypeptide of the present inventionmay have. Further, examples of the partial peptide of the presentinvention, similar to the polypeptide of the present invention describedabove, include a peptide wherein an amino group of a methionine residueat the N-terminus is protected with a protecting group; a peptidewherein a glutamyl residue at the N-terminus which is generated by invivo cleavage is pyroglutamated; a peptide wherein a substituent on theside chain of intramolecular amino acids is protected with anappropriate protecting group; a complex peptide, such as a so-calledglycopeptide formed by the binding of sugar chain to a peptide, or thelike. The partial peptide of the present invention may be used as, forexample, a reagent, reference or standard materials for experiments, oran immunogen or a portion thereof.

Particularly preferred salts of the polypeptide of the present inventionor the partial peptide are physiologically acceptable acid-added salts.Examples of such salts that are used herein include a salt formed withan inorganic acid (for example, hydrochloric acid, phosphoric acid,hydrobromic acid and sulfuric acid), and a salt formed with an organicacid (for example, acetic acid, formic acid, propionic acid, fumaricacid, maleic acid, succinic acid, tartaric acid, citric acid, malicacid, oxalic acid, benzoic acid, methane sulfonic acid andbenzenesulfonic acid).

The polypeptide of the present invention, the partial peptide thereof orsalts thereof, or amides thereof can be prepared by a chemical synthesismethod known in the art.

For example, amino acids whose α-amino groups and side chain functionalgroups are appropriately protected are condensed on resin (which iscommercially available resin for protein synthesis) in accordance withthe sequence of a target polypeptide, according to various condensationmethods known in the art. Various protecting groups are then removedsimultaneously with cleavage of the polypeptide from the resin at theend of reaction. Further, reaction for forming an intramoleculardisulfide linkage is conducted in a highly diluted solution, therebyobtaining a target polypeptide, the partial peptide thereof or amidesthereof. Examples of activation reagents that may be used to condensethe above protected amino acids include those that may be used forpolypeptide synthesis and are represented by carbodiimides, such as DCC,N,N′-diisopropylcarbodiimide and N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide. For activation by such reagents, both protected aminoacids and a racemization-suppressing additive (for example, HOBt orHOOBt) are directly added to the resin; or protected amino acids may beactivated in advance as corresponding acid anhydride, HOBt ester orHOOBt ester, and then added to the resin.

Solvents used for the activation of protected amino acids andcondensation with resin may be appropriately selected from solventsknown in the art as applicable to polypeptide condensation reaction,such as acid amides, halogenated hydrocarbons, alcohols, sulfoxides andethers. A reaction temperature is appropriately selected from a knownrange that may be used for reaction of polypeptide linkage formation.Activated amino acid derivatives are normally used in an amount of 1.5to 4-fold excess. When condensation is found to be insufficient as aresult of a test using ninhydrin reaction, condensation reaction withouteliminating protecting groups will be repeated for sufficientcondensation. When condensation is found to be still insufficient,unreacted amino acids are acetylated using acetic anhydride oracetylimidazole so as not to affect the subsequent reaction.

Protecting groups normally employed in the art may be used for each ofamino groups, carboxyl groups and serine hydroxyl groups of rawmaterials.

The protection of functional groups that should not involve the reactionof raw materials, protecting groups, and the elimination of theprotecting groups, and the activation of functional groups that involvereaction and the like may be appropriately selected from known groups orperformed by known measures.

The partial peptide of the present invention or a salt thereof may beproduced according to a peptide synthesis method known in the art, or bycleaving the polypeptide or the present invention with an appropriatepeptidase. For example, the peptide synthesis method may be either asolid-phase synthesis method or a liquid phase synthesis method. Exampleof the known condensation method and the method of elimination ofprotecting groups are described in Nobuo IZUMIYA et al., Basics andExperiment for Peptide Synthesis, Maruzen (1975); Haruaki YAJIMA andShunpei SAKAKIBARA, Experiment Course for Biochemistry 1, ProteinChemistry IV, 205 (1977); and Development of Pharmaceutical Preparation,vol. 14, Peptide Synthesis, under the editorship of Haruaki YAJIMA,Hirokawa Publishing Co.

After reaction, the partial peptide of the present invention may bepurified and isolated by an appropriate combination of known methods,such as solvent extraction, distillation, column chromatography, liquidchromatography, and re-crystallization. When the partial peptideobtained by the above methods is a free one, it can be converted to anappropriate salt by a known method. Conversely, when the peptide isobtained as a salt, it can be converted to a free one by a known method.

The antibody against the polypeptide of the present invention, thepartial peptide thereof or salts thereof may be either a polyclonal or amonoclonal antibody, so far as it can recognize these substances. Theantibody against the polypeptide of the present invention, the partialpeptide thereof or salts thereof may be produced using as an antigen thepolypeptide of the present invention or the partial peptide thereofaccording to a known method for producing antibodies or anti-serum.

The antibody of the present invention may be used to detect thepolypeptide of the present invention and the like which are present in aspecimen, such as body fluid, tissues or the like. In addition, theantibody may be used for preparing an antibody column to be used forpurifying these substances; detecting the polypeptide of the presentinvention in each fraction upon purification; analyzing the behavior ofthe polypeptide of the present invention within the cells of a specimen;and the like.

The use of the DNA, the polypeptide and the antibody of the presentinvention will be further described below.

Abnormalities (of the gene) in DNA or mRNA encoding the polypeptide ofthe present invention or the partial peptide thereof may be detectedusing as a probe the DNA of the present invention, the antisense DNA ofthe DNA of the present invention, or a gene construct comprising theseDNAs.

The DNA, the antisense DNA or the gene construct of the presentinvention are useful as a genetic diagnostic agent for, for example,damages, mutation or hypoexpression in the DNA or mRNA, and an increaseor hyperexpression of the DNA or mRNA. The above gene diagnosis usingthe DNA of the present invention may be performed by, for example, aknown northern hybridization or a PCR-SSCP method (Genomics, 5:874-879(1989), Proc. Natl. Acad. Sci. USA, 86:2766-2770(1989)).

Moreover, for patients whose function does not works in vivo because ofabnormalities or deletion in the DNA or the gene of the presentinvention, or because the expression amount of the DNA or the gene ofthe present invention is reduced, it is effective that the DNA or thegene construct of the present invention is introduced for expressioninto the bodies of the patients by gene therapy using as vehiclesappropriate vectors, such as retrovirus vectors, adenovirus vectors andadenovirus-associated virus vectors according to known techniques.Further, when patients cannot exert normal functions because of theincreased expression amount, introduction of antisense may be effective.

The DNA, the antisense DNA of the present invention, or the geneconstruct thereof may be administered alone, or in combination with anadjuvant to promote uptake using a gene gun or a catheter, such as ahydrogel catheter.

In another example, injection of the polypeptide of the presentinvention or the like into patients with the above diseases also enablesthe polypeptide of the present invention or the like to exert itsfunction in the patients.

Furthermore, the antibody of the present invention may be used forquantitatively determining the polypeptide of the present invention in atest liquid by a known method. Specifically, the antibody of the presentinvention may be used for quantitative determination by a sandwichimmunoassay using monoclonal antibodies, detection by tissue staining,and the like, by which, for example, diseases that involve thepolypeptide of the present invention or the like may be diagnosed.

For these purposes, an antibody molecule itself may be used, or theantibody molecules F(ab′)₂, Fab′ or Fab fractions may be used.Quantitative determination methods for the polypeptide of the presentinvention using the antibody of the present invention are notspecifically limited. Any measurement method can be used, so far as itinvolves detecting the amount of antibodies, antigens orantibody-antigen complexes corresponding to the amount of antigens (forexample, protein amount) in a test liquid by chemical or physical means,and calculating with a calibration curve which has been prepared using astandardized solution containing a known amount of antigens. Forexample, nephrometry, competitive assay, immunometric assay and sandwichassay are preferably used. Examples of a labeling agent that may be usedin a measurement method using a labeling substance include a substanceknown in the art, such as radioisotopes, enzymes, fluorescent materialsand light-emitting materials.

Details about the general technical procedures concerning thesemeasurement and detection methods may be referred to in a review,reference book or the like, such as Radioimmunoassay 2 edited by HiroshiIRIE, (Kodansha, issued in 1979); Enzyme Immunoassay edited by EijiISHIKAWA et al., (3^(rd) edition; Igakushoin, issued in 1987); andMethods in Enzymology (issued by Academic Press), vol. 70,“Immunochemical Techniques (Part A)”, vol. 73, “ImmunochemicalTechniques (Part B)”, vol. 74, “Immunochemical Techniques (Part C)”,vol. 84, “Immunochemical Techniques (Part D: Selected Immunoassays)”,vol. 92, “Immunochemical Techniques (Part E: Monoclonal Antibodies andGeneral Immunoassay Methods)”, and vol. 121, Immunochemical Techniques(Part I: Hybridoma Technology and Monoclonal Antibodies)”.

Moreover, the DNA chip prepared by arraying the DNA of the presentinvention is useful in detecting mutations and polymorphism of the DNAof the present invention, and monitoring the DNA dynamics. Regarding DNAarray, which is a type of DNA chip, see “DNA microarray and Current PCRmethod” (a supplementary volume of Cell Technology, Genome ScienceSeries 1, under the editorship of Masaaki MURAMATSU and Hiroyuki NABA,1^(st) edition, issued on Mar. 16, 2000) and the like.

Further, the polypeptide chip prepared by arraying the polypeptide ofthe present invention is a strong tool for functional analysis on theexpression, interaction and posttranslational modification of thepolypeptides of the present invention, and for identification andpurification of proteins.

The antibody chip prepared by arraying antibodies against thepolypeptides of the present invention is very useful in analyzing thecorrelation between the polypeptides of the present invention anddiseases, disorders, or other physiological phenomena.

Methods and materials for preparing those chips are well known for thoseskilled in the art.

Furthermore, the polypeptides of the present invention or the like areuseful as reagents for screening compounds which interact specificallywith these substances. More specifically, the present invention providesa method for screening compounds specifically interact with thepolypeptide of the present invention, a partial peptide thereof or saltsthereof by using these substances or the salts thereof; and provides thescreening kit therefor.

Compounds or salts thereof that are identified by using the screeningmethod or the screening kit of the present invention interact with thepolypeptide of the present invention or the like so as to, for example,regulate, inhibit, promote or antagonize the biological activity of thepolypeptide of the present invention or the like. The compound or thesalt thereof may directly act on the activity of the polypeptide of thepresent invention or the like, or indirectly act on the activity of thepolypeptide of the present invention or the like by acting on theexpression of the polypeptide of the present invention or the like. Anexample of the salt of the compound that is used herein is apharmaceutically acceptable salt. Specific examples of such saltsinclude a salt formed with inorganic base, a salt formed with organicbase, a salt formed with inorganic acid, a salt formed with organicacid, and a salt formed with basic or acidic amino acid. Compounds thatinhibit the biological activity of the polypeptide of the presentinvention or the like may also be used as pharmaceutical preparations,such as therapeutic agents and preventive agents for each of theabove-mentioned diseases.

The abbreviation for a base and amino acid is shown in the presentspecification in accordance with IUPAC-IUB Commision on BiochemicalNomencalture or conventional methods, and an optical isomer of the aminoacid, if any, means its L-isomer unless otherwise noted.

EXAMPLES

The present invention will by further explained by the followingexamples, which do not limit the scope of the present invention. Thegenetic procedures in the examples are done in accordance with thosedescribed in Current protocols in molecular biology (edited by FrederickM. Ausubel et al., 1987).

(1) Construction of cDNA Library Derived from Human Adult Whole Brainand Human Fetal Whole Brain

A double-stranded cDNA was synthesized by SuperScriptII reversetranscriptase kit (Invitrogen Co.) with the use of an oligonucleotidehaving NotI site (GACTAGTTCTAGATCGCGAGCGGCCGCCC(T)₁₅) (Invitrogen Co.)as a primer, and mRNA derived from human adult whole brain and humanfetal whole brain (Clontech Co.) as a template. An adapter having SalIsite (Invitrogen Co.) was ligated with the resulting cDNAs. Afterdigestion with NotI, the cDNAs were subjected to electrophoresis on alow-melting agarose of 1% to purifiy cDNA fragments with 3 kb or more.

The thus purified cDNA fragments were ligated with pBluescript IISK+plasmid treated with SalI-NotI restriction enzymes. The resultingrecombinant plasmids were introduced into E. coli DH10B strain(Invitrogen Co.) by an electroporation method.

(2) Screening

The terminal nucleotide sequences of randomly selected clones weredetermined, and the homology search was done on nr database (allGenBank+EMBL+DDBJ+PDB sequences, but no EST, STS, GSS or phase 0.1 or 2HTGS seqeunces) with the use of the resulting sequences as a query inaccordance with homology search program BLASTN2.2.1 (Altshul, StephenF., Thomas L. Madden, Alejandro A., Schaffer, Jinghui Zhang, ZhengZhang, Webb Miller, and David J. Lipman (1997), “Gapped BLAST andPSI-BLAST: a new generation of protein search programs.”, Nucleic acidsRes. 25:3389-3402). As a result, the terminal sequences of 3′- and5′-ends of a gene having no homologous gene, i.e., a novel gene werealigned with human genomic sequence (ftp://ncbi.nlm.nih.gov/genomes/Hsapiens/) with the use of homology search program BLASTN2.2.1.

Genes were picked up from a genome region inserted between them by theuse of Genscan program (computer software for predicting a gene fromgenome sequences) (Burge, C. and Karlin, S. 1987, Prediction of completegene structures in human genomic DNA, J. Mol. Biol., 268, 78-94).Homology search was done on mergedb, which had been prepared bycombining human cDNA sequences determined by KAZUSA DNA Institute andHomo sapiens database of GenBank (except EST and genome) withoutoverlapping data, with the use of the selected genes as a query inaccordance with homology search program BLASTN2.2.1. When a novellong-ORF gene (with 1,200 bp or more of cds according to the predictionby Genscan) was found, the full-length sequences of its 5′- and 3′-endswere determined.

Determination of the nucleotide sequence was carried out by means of aDNA sequencer (ABI PRISM377) and a reaction kit manufactured by PEApplied Bio System Co. Most of the sequences were determined by aditerminator method on shotgun clones, and parts of them were determinedby a primer-walking method with the use of oligonucleotides that weresynthesized based on the thus determined nucleotide sequences.

The novel DNAs or genes were screened in the above ways. As a result, aclone fj03204 was found.

(3) Homology Search of the Present DNA

The homology search of the whole nucleotide sequence thus determined wasdone on the known nr data in accordance with homology search programBLASTP 2.2.1 (Altshul, Stephen F., Thomas L. Madden, Alejandro A.,Schaffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman(1997), “Gapped BLAST and PSI-BLAST: a new generation of protein searchprograms.”, Nucleic acids Res. 25:3389-3402). As a result, it isrevealed that the present DNA has homology to a gene shown in Table 1.Table 1 shows information about the gene (homologous gene) such as itsname, data base ID, species, length of protein and references. TABLE 1Homologous Length of gene Data base ID Protein Species ReferenceQuiescin Q6 gi | 13325075 747 Human Genomics(1998), 54 (3); 460-8Quiescin Q6 gi | 16758172 570 Rat J.B.C.(2001), 276(17); 13830-7Quiescin Q6 gi | 12963609 568 Mouse Genome Res. 2000, 10(10); 1617-30

Table 2 summarizes a variety of data concerning homology between the DNAor the genes of the present invention and each homologous gene listed inTable 1.

The meaning of each item in Table 2 is as follows:

“Score”: the higher this value is, the higher credibility is;

“E-value”: the closer this value comes to “0”, the higher credibilitybecome;

“Homology”: the percentage of identical amino acids in the homologousregion; and

“Homology region percentage (%)”: the percentage of the homologousregion in the homologous gene. TABLE 2 Homology Value Homologous regionPercentage of Clone Homologous gene the homology from to Species from toScore E-value Homology region 34 629 Human 12 613 464 e−135 41%(252/611)81% 23 583 Rat 6 559 474 e−132 44%(246/565) 97% 23 588 Mouse 6 565 483e−1129 45%(257/572) 99%

(4) Search of Domains

Using as queries the amino acid sequence encoded by DNAs contained inthe clones, functional domains were searched with a search toolcontained in Pfam 7.6 (Pfam HMM ver. 2.1 Search (HMMPFAM), Sonnhammer,E. L. L., Eddy, S. R., Birney, E., Bateman, A., and Durbin, R. (1998)“Pfam: multiple sequence alignments and HMM-profiles of proteindomains”, Nucleic Acids Res. 26:320-322).

Further, transmembrane domains were searched with a prediction programfor membrane proteins, the SOSUI system (ver. 1.0/10, March, 1996)(Takatsugu Hirokawa, Seah Boon-Chieng and Shigeki Mitaku, SOSUI:Classification and Secondary Structure Prediction System for MembraneProteins), Bioinformatics (formerly CABIOS) 1998 May; 14(4): 378-379).

Table 3 shows the detected functional domains and transmembrane domainsfor each clone.

The meaning of each item in Table 3 is as follows:

Functional domain: a domain detected by Pfam or SOSUI

Starting point (From): an amino acid position as a starting point of afunctional domain

End point (To): an amino acid position as an end point of a functionaldomain

Score (Pfam only): the higher the value, the higher the reliability

Exp (Pfam only): the closer the value to 0, the higher the reliabilityTABLE 3 fj03204 Human Quiescin Functional Functional domain From ToScore Exp domain From To Score Exp sosui 29 51 28.7 6.1e−06 Thioredoxin39 155 52.5 9e−12 Thioredoxin 60 177 sosui 654 676 Rat Quiescin MouseQuiescin Functional Functional domain From To Score Exp domain From ToScore Exp sosui 12 34 49.0 1e−10 sosui 11 33 45.8 9.9e−10 Thioredoxin 42162 Thioredoxin 42 162 sosui 63 85 sosui 63 85

(5) Expression Site

Expressions in the tissues and the sites of the brain were examined byRT-PCR ELISA (Nagase, T., Ishikawa, K., Suyama, M., Kikuno, R.,Miyajima, N., Tanaka, A., Kotani, H., Nomura, N. and Ohara, O.Prediction of the coding sequences of unidentified human genes. XI. Thecomplete sequences of 100 new cDNA clones from brain which code forlarge proteins in vitro. DNA Res. 1998 Oct. 30; 5(5): 277-86). Table 4shows the result.

The expression amount (unit (fg) per ng of poly(A)+ RNA) is representedby + for less than 0.1; ++ for more than 0.1, less than 100; and +++ formore than 100.

Table 5 shows the complete notation of each tissue and site of thebrain. TABLE 4 Adult Site of Clone Tissue the brain name He Br Lu Li SmKi Pa Sp Te Ov Am Co fj03204 +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ ++++++ Adult Embryo Clone Site of the brain Tissue name Ov Co Ce Ca Hi NiNu Th Sp Li Br fj03204 +++ +++ +++ +++ ++ +++ +++ +++ +++ +++ +++

TABLE 5 Abbreviated notation Complete notation Tissue Br Brain He HeartKi Kidney Li Liver Lu Lung Ov Ovary Pa Pancreas Sm Skeletal muscle SpSpleen Te Testis Site of the brain Am Amygdala Ca Caudate nucleus CeCerebellum Co Corpus callosum Hi Hippocampus Ni Substantia nigra NuSubthalamic nucleus Th Thalamus Sp Spinal cord

(6) Location on Chromosome

Using the DNA base sequence of the clones as queries, theabove-mentioned analysis program BLASTN 2.2.1 (“Gapped BLAST andPSI-BLAST: a new generation of protein database search programs”) wasrun on the human genome sequences in the library of known sequences,Genbank release 122 and 123. Additionally, using the homology searchprogram BLASTN 2.2.1, the DNA sequence of the clone was aligned with thelibrary of clones encoding human genome(ftp://ncbi.nlm.nih.gov/genomes/H_sapiens/). As a result, it has beenrevealed that the present DNA is located on chromosome 2 (2q21).

(7) Expression of the Protein Encoded by the Present Gene

A gene product was expressed from the cDNA clone fj03204 with the use ofthe transcription and translation system in vitro (Promega Co., TNT T7Quick Coupled Transcription/Translation System cat.no.L1107).

The product incorporated with ³⁵S-labeled methionine was subjected toSDS-PAGE (12.5%). After drying of a gel, autoradiography was done withthe use of BAS2000 (Fuji film) system to detect the gene product of theclone fj03204. As a result, a band, which was presumed to be atranscription/translation product of the clone fj03204, was observed ata point corresponding to a marker with 77 kDa.

As a molecular weight of the protein encoded by the fj03204 consistingof 698 amino acids from a first methionine is presumed to be about77,350 Da, the presumed molecular weight was coincided well with theabove result.

(8) Function of the Present Gene

The data concerning homology, information about the homologous genes,domains, expression sites and location on chromosome have revealed thefollowings:

The searches of homology and functional domains clarified that thepresent DNA or gene has about 40% homology with Quiescin Q6 Gene family,and has a thioredoxin domain at its N-terminus. Further, as seen fromalignment with the amino acid sequences of Quiescin Q6 Gene (QSCN 6)disclosed in Non-patent documents 1 and 2, the present DNA or gene hasERV1 of yeast at its C-terminus, i.e., amino acids 405-539 in SEQ IDNo.1. These facts suggest that the present DNA or gene belongs toQuiescn Q6 Gene family (QSCN 6) as disclosed in Non-patent documents 1and 2.

As described in Non-patent documents 1 and 2, ERV1 gene is essential foroxidative phosphorylation and asexual reproduction of yeast (Lisowsky,T. (1992), Mol. Gen. Genet., 232:58-64), and play an important role inmaintenance and cell division cycle of yeast mitochondria genome(Lisowsky, T. (1994), Curr. Genet., 26:15-20).

QSCN6 is lowly expressed in a fibroblast that is actively growing, andhighly expressed in that in quiescent phase. While the expression ofmRNA of QSCN6 is strongly induced at the time when the fibroblast beginsto shift from growth phase into quiescent phase, it is stronglyinhibited in a transformed fibroblast (Coppock D. L. et al., (1993) CellGrowth Differ., 4:483-493).

Some of the other genes belonging to the above group are a component ofextracellular matrix (ECM), which plays not only a structural role in acell but also a functional role such as suppression of tumor orregulation of growth.

From the above information, it seems that QSCN6 may possibly play a rolein cell adhesion. It may further function during the entering process ofa normal cell into an irreversible quiescent phase. It is also suggestedthat the inhibition of this gene may play a part in canceration(malignant alternation). Further it seems that QSCN6 may play a role incontrol of cell growth and redox state.

Accordingly, those skilled in the art may reasonably presume that thepresent DNA or gene has function that is deeply related to diseasesinvolved aging or cancer.

As seen form the base sequence of clone fj03204 represented by SEQ IDNo.1, the surrounding base sequence (AACATGG) of its translationinitiation codon coincides well with Kozak consensus sequence (ACCATGG).Furthermore, while QSCN6 protein exists in matrix of yeast mitochondriaas described in Non-patent document 2, a signal sequence necessary forsecretion (the region detected by Sosui program) is found at theN-terminus of the clone fj03204. This fact means that the clone fj03204encodes the gene with a full length.

INDUSTRIAL APPLICABILITY

Cell growth is a dynamic process that is determined by the ratio among afraction of cells in growth phase, a fraction of cells in quiescentphase, and a fraction of dead cells. The regulation of transition fromthe growth phase to the quiescent phase is an important step in thewhole regulation of a growth process. The inhibition of a normaltransition into the quiescent phase is characteristic in cancer andother proliferative diseases.

It is expected therefore that the present invention contributes todiagnosis and treatment of the diseases involved aging or cancer.

Further, a single nucleotide polymorphism, SNP, which is a change in onebase (nucleotide) among individuals in the DNA or the gene of thepresent invention, can be found by performing PCR using synthetic DNAprimers prepared based on the nucleotide sequence of the DNA or the geneof the present invention or a part thereof, and using chromosome DNAextracted from human blood or tissue so as to determine the nucleotidesequence of the product. Therefore, individual constitution or the likecan be predicted, which enables the development of a pharmaceuticalpreparation suitable for each individual.

Further, when ortholog (homolog, counterpart) genes for the DNA or thegene of the present invention in model organisms, such as mice, areisolated with cross hybridization, for example, these genes are knockedout to produce human disease model animals, so that the causative geneswhich cause human diseases can be searched and identified.

Novel DNAs or genes obtained by the present invention are assembled on aso-called DNA chip, and then probes prepared using blood or tissuederived from cancer patients or patients with diseases that relate tothe brain, such as mental disease, or as a control using blood or tissuefrom healthy individuals are hybridized to the chip, so that the chipcan be applied to diagnosis and treatment for the diseases.

Moreover, antibody chip, on which the antibodies against thepolypeptides of the present invention are thoroughly prepared andarrayed, can be applied to diagnosis, treatment of diseases and the likethrough proteome analysis, such as detection of a difference inexpression amount of a protein between a patient and a healthyindividual.

Furthermore, the DNAs or the gene constructs of the present inventioncan be used as an active ingredient of vaccine.

The present application asserts priority based on the specification ofJapanese Patent Application No. 2002-205915 and includes by referenceall of the contents as disclosed in the specification.

1. A DNA comprising a base sequence encoding the following polypeptide(a) or (b): (a) a polypeptide consisting of an amino acid sequence whichis identical or substantially identical with the amino acid sequencerepresented by SEQ ID No.1, (b) a polypeptide consisting of an aminoacid sequence represented by SEQ ID No.1 in which part of amino acidsare deleted, substituted or added, and having substantially the samebiological activity as the function of the polypeptide (a).
 2. A DNAhybridizing with a DNA having a base sequence complementary to the DNAof claim 1 under stringent conditions, and having substantially the samebiological activity as the function of the above polypeptide (a) ofclaim
 1. 3. A gene construct comprising the DNA of claim 1 or
 2. 4. Apolypeptide (a) or (b): (a) a polypeptide consisting of an amino acidsequence which is identical or substantially identical with the aminoacid sequence represented by SEQ ID No.1, (b) a polypeptide consistingof an amino acid sequence represented by SEQ ID No.1 in which part ofamino acids are deleted, substituted or added, and having substantiallythe same biological activity as the function of the polypeptide (a). 5.A recombinant polypeptide encoded by the gene construct of claim
 3. 6.An antibody against the polypeptide of claim 4 or
 5. 7. A DNA tip onwhich the DNAs of claim 1 or 2 are arrayed.
 8. A polypeptide tip onwhich the polypeptides of claim 4 or 5 are arrayed.
 9. An antibody tipon which the antibodies of claim 6 are arrayed.
 10. An antisenseoligonucleotide to the DNA of claim 1 or 2.